1. Field of the Invention
The present invention relates to techniques for detection and localization of nucleic acid and, more particularly, to the use of enzyme catalyzed chromogenic compositions in such detection and localization.
2. Description of Related Art
In-situ hybridization (ISH) techniques are an important tool for detection of nucleic acid sequences, i.e., both DNA and RNA. Unique nucleic acid sequences occupy precise positions in chromosomes, cells and tissues and in-situ hybridization allows the presence, absence and/or amplification status of such sequences to be determined without major disruption of the sequences.
It is known that certain nucleic acid sequences are associated pathologic conditions in living organisms. For example, the presence of certain genes and viral nucleic acids have been implicated in precancerous and cancerous pathology. Genetic diseases are also diagnosed by determining the presence, absence or number of copies of nucleic acids. Several genetic markers have been associated with poor prognosis in patients with various cancers. Infectious microorganisms, particularly intracellular ones, contain nucleic acid sequences which are also detected to diagnose disease and monitor therapy.
Accumulation of alterations in both cellular oncogenes and tumor suppressor genes has been associated with human tumorigenesis. Gene amplification has been associated with certain aggressive forms of human cancer and has been used as a prognostic parameter in the clinical analysis of certain malignancies. Presence or absence of nucleic acid amplification can also be used to indicate treatment in certain cancers of disease states. Cellular oncogene amplification of the HER-2/neu oncogene has been shown to play an important part in the pathogenesis and prognosis of various solid tumors including breast cancer. See, e.g., Battifora et al., Modern Pathol, 4:466–474 (1991) and Press et al., Cancer Res., 53:4690–4970 (1993).
Loss and/or mutation of tumor suppressor genes is also indicative of certain cancers and certain stages of cancers. The loss of p53 is classically found in many solid tumors. In such a situation a cell usually has only one mutated copy of the gene, the other copy being lost due to aneuploidy. Loss of both copies is also found.
Human Papilloma virus (HPV) is a common sexually transmitted viral disease. There are at least 70 distinct types of HPV. Some HPV types found in genital lesions have been implicated in cervical precancers and cancers (for example, types 16, 18, 31, 33 and 35) while other types are relatively benign (Types 6, 11, 42, 43 and 44).
Currently, Pap smears are performed yearly on women to check for the presence of atypical or cancerous cells. Roughly 90% of all Pap smears are normal, 3% are unequivocally dysplastic, and 7% are squamous atypias (ASCUS) or low grade squamous intraepithelial lesions (LSIL). The ASCUS and LSIL diagnoses present the doctor and patient with multiple choices for treatment. The ability to accurately test these patients for high risk type HPV presence would provide further information on the best course of therapy. For example, the presence of a low risk HPV type may indicate no further action except perhaps more frequent Pap smears. A high risk HPV type presence would indicate a more aggressive approach.
Other viral diseases are also frequently difficult to detect or distinguish clinically. Examples include Epstein-Barr virus (EBV), cytomegalovirus (CMV), hepatitis viruses, etc. Nucleic acid based detection systems performed in-situ for these viruses is also desirable.
At least four types of nucleic acid probes are commonly used for in-situ hybridization. These include double stranded DNA (dsDNA) probes, single stranded DNA probes (ssDNA), single-stranded RNA probes (ssRNA), and oligonucleotide probes. The production and application of a large variety of DNA and RNA probes has been made possible through the availability of many molecular cloning techniques including plasmid, phage P1, cosmid, and yeast artificial chromosome (YAC) cloning procedures, cell hybrid technology, chromosome sorting and dissection techniques, and amplification techniques such as the polymerase chain reaction (PCR). Additionally, the use of DNA synthesizers can permit oligonucleotides to be custom designed and chemically synthesized. Different target sequences such as specific genomes, chromosomes, repetitive and unique sequences, microsatellites, mitochondrial nucleic acids, mRNA, or microbial (viral) nucleic acids may be identified depending on the selection of probe used in the ISH procedure. Nucleic acid probes may be labeled by conjugation to a marker to create a detectible probe hybridization site.
A variety of detection systems have been developed which are based on ligands which bind to a probe either directly or indirectly and markers or labels which allow visualization of the probe and hence, the site where the probe has hybridized. Radioactive labels or non-radioactive fluorescent labels have been employed as such markers or labels either directly linked to the probe or attached through secondary means such as antibodies. Although radioactive labels are effective, they are associated with radioactive toxicity and environmental concerns. Fluorescent non-radioactive detection protocols provide several advantages for in-situ hybridization, including easy and rapid detection, high sensitivity with low endogenous background, high resolution, multiple-target analysis with different fluorochromes, and the possibility to quantitate signal. Unfortunately, the signal generated by fluorescent markers typically fades over time. Upon exposure to light and autofluorescence of the tissue sample may mask the presence of a target signal. Additionally, the cost and availability of fluorescent microscopy equipment and trained personnel is greater than conventional brightfield microscopy.
Alternatively, enzyme systems have been used for detection of nucleic acid target sequences. Enzymes such as horseradish peroxidase or alkaline phosphatase can be chemically conjugated to proteins, antibodies, avidin, streptavidin, biotin, Fc-binding proteins such as protein A or G for use in hapten interactions, or directly to the nucleic acid probes. Certain enzymes interact with chromogen substrate solutions to produce distinctly colored products which are capable of being visualized directly through brightfield microscopy. This permits the localization of hybridization sites through enzyme precipitation reactions. Some advantages of cytochemical detection with enzymes include the stability of the precipitate, indicating permanent storage of cell preparations, and the use of a standard brightfield microscope in a setting where routine analysis is performed.
Oxidoreductases are enzymes which catalyze the oxidation of various substrates, and are well suited for the preparation of enzyme-conjugates due to their excellent stability and their ability to yield chromogenic products. Peroxidases have been widely used as a label for antibodies and ligands, such as avidin and streptavidin, in immunoassay systems. Peroxidases catalyze the hydrogen peroxide oxidation of certain electron donors by transferring electrons from the donor to the peroxide and resulting in formation of a colored product and water.
A number of different chromogens have been used with enzyme-linked immunoassays (ELISA). See, e.g., U.S. Pat. No. 4,962,029. A number of other different enzymes such as kinases and phosphatases catalyze the addition and removal of phosphate moieties. These enzymes have also been used in various immunoassays.
TMB has reportedly been used in detection of repetitive DNA sequences in Speel et al., Rapid Bright-Field Detection of Oligonucleotide Primed In-Situ (PRINS)—Labeled DNA in Chromosome Preparations and Frozen Tissue Sections, Biotechniques, 20:226–234 (February 1996). The PRINS procedure involves placing haptens at a target site using enzymes to incorporate labeled nucleotides by elongating unlabeled primers. More specifically, unlabeled DNA primer is annealed to its complementary target sequence in-situ. The primer serves as an initiation site for chain elongation using DNA polymers and fluorochrome-, biotin-, or digoxigenin-labeled nucleotides. The labeled DNA chain is then detected directly by fluorescence microscopy or indirectly by fluorochrome-conjugated avidin or antibody molecules. Speel et al. describes localization of DNA target sequences using PRINS and colored precipitates of horseradish peroxidase-diaminobenzidine (brown color), alkaline phosphatase-Fast Red (red color) and horseradish peroxidase-tetramethylbenzidine (green color). Results were evaluated using bright-field microscopy.
In Speel et al., A Novel Triple-color Detection Procedure for Bright-field Microscopy, Combining In-Situ Hybridization with Immuno Chemistry, J. Hist. Cyt., vol. 47, No. 10, pp. 1299–1307 (1994), a peroxidase-TMB product was detected using in-situ hybridization techniques. The system described by either Speel et al. publication above was used only for the detection of satellite or repetitive DNA. However, the sensitivity required for detecting such multiple copy sequences is much lower than the sensitivity required for detection of unique copy sequences. If the sensitivity of techniques using labeled nucleic acids and their detection systems can be increased to allow detection of unique copy sequences, more accurate results could be obtained for detection of such sequences in diagnosis and prognostication of cancer or other disease states.
A number of patents have proposed various colormetric determinations of hybridization. These include U.S. Pat. Nos. 5,851,764, 5,846,728, 5,525,465, 5,677,440 and 5,474,916. However, none of these were able to distinguish single gene copies in-situ.